The field of the invention is anti-viral agents.
Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering effectiveness.
HIV-1 is a member of the lentivirus family of retroviruses. Upon infection with the virus, individuals exhibit a variable onset of AIDS-related diseases (Levy, 1993.). Recent studies have shown that even in the midst of the clinically latent period, there is no virological latency in the infected individuals (Pantaleo et al., 1993; Embretson et al., 1993). Given this scenario, it has been suggested that inhibitors of virus replication will be of immense value in the onset and control of AIDS-related diseases in persons infected with HIV-1 (Ridky et al., 1995; Miller et al., 1996; Mellors et al., 1996; Crowe et al., 1996).
The HIV-1 life cycle shares several features common to all retroviruses. These features include virus attachment to a specific receptor, penetration, uncoating, reverse transcription, translocation of viral DNA from the cytoplasm to the nucleus, integration, expression of viral proteins, assembly, and maturation of virus particles (1). Virally encoded enzymatic activities, which are essential for the processes associated with virus infection, have all been used as targets for developing agents that interfere with the virus replication (2-4). Unfortunately, the use of such antiviral agents has also resulted in the emergence of drug-resistant viruses (5-7). In comparison to the monotherapy, combination therapy involving multiple inhibitors has been shown to be effective (8-11). The emergence of drug-resistant viruses, however, will remain a problem with the continued use of antiviral agents to target viral enzymes. Hence, alternative strategies to contain HIV-1 replication are warranted. Toward this goal, an approach to generate a novel anti-HIV-1 agent from within the virus has been considered.
Unlike the simple retroviruses, the HIV-1 genome contains six auxiliary genes in addition to the gag, pol and env common to all retroviruses. The processes associated with virus infection are carried out by different viral gene products, which makes these proteins potential targets for antiretroviral therapy. These processes include: a) binding to a receptor and virus internalization, b) reverse transcription and transport of viral DNA to the nucleus for integration, c) expression of viral proteins and d) assembly and releases of viral particles from infected cells (Levy, 1993).
Among the auxiliary gene products of HIV-1, vpr, vif, and nef have been shown to be associated with virus particles to a varying extent (12-16). The virion-associated protein Vpr has been an intensive area of interest with respect to understanding the role of Vpr in virus infection. Vpr coding sequences (96 aa) are found to overlap Vif at the 5' end and Tat at the 3' end (17). Characteristic features of Vpr include virion incorporation, cell cycle arrest at the G.sub.2 stage, nuclear localization, participation in transport of the prointegration complex, demonstration of cation channel activity, and interaction with several candidate cellular proteins (18-28). Additionally, work from our laboratory and others has shown that Vpr is essential for optimum infection of macrophages (29, 30). Mutational analysis of Vpr has revealed the presence of critical domains needed for its virion incorporation and the importance of the predicted helical domain (amino acids 17-34) in such an event (22, 31-38). The virion specificity and abundance of Vpr in viral particles provide avenues for localizing antiviral agents to progeny virus, giving the ability to interfere with the assembly, maturation, and infectivity of HIV-1.
Upon initial synthesis as a polyprotein precursor, the HIV-1 aspartyl protease has the unique ability to autocatalyze its own cleavage from the Pr160 polyprotein precursor. After its release, the protease is then able to catalyze the cleavage at other sites generating the mature Gag protein, p17, p24, p7, and p6 and the reverse transcriptase (RT) and integrase enzymes. The specific cleavage sites between the proteins in the polyprotein precursor recognized by HIV-1 protease are highly conserved among viral isolates (39).
In the present invention, in order to generate an effective anti-HIV-1 agent from within the virus, we have combined the protease cleavage site residues found in the Gag and Gag-Pol precursor proteins and the virion-specific feature of Vpr. The rationale for this approach is that an inappropriate presentation of chimeric Vpr (Vpr-C) proteins containing protease cleavage signal sequences might interfere with the processing of bona fide viral precursor polyproteins leading to the generation of incompletely processed noninfectious virus particles (2). The differential amount of Gag, Gag-Pol, and Vpr proteins present in the virus particles supports the feasibility of such an approach. The results demonstrate that the above strategy is effective in interfering with HIV-1 virus replication.
As regards a second aspect of the invention, it is relevant that HIV-1 PR is an obligatory homodimer, with the active site made from two monomers containing DTG amino acid residues brought into proximity through regions in the NH.sub.2 and COOH termini of the PR monomers, known as the dimer interface (Babe et al., 1991). The dimer interface region of ribonucleotide reductase and DNA polymerase from herpes simplex virus and HIV-1 RT have been investigated as potential targets for developing antiviral agents (Dutia et al., 1986; Cohen et al., 1986; Divita et al., 1994; Digard et al., 1995). It has been suggested that peptides corresponding to the dimer interface structure between the HIV-1 PR subunits may interfere with the generation of an enzymatically active molecule. It is estimated that the C-terminal 4 residues of PR contribute up to 50% of the intersubunit interaction in the dimer (Weber, 1990). A synthetic peptide corresponding to this domain showed dissociative inhibition of HIVI PR in vitro and a weak inhibition of viral replication in cell cultures (Zhang et al., 1991; Schramm et al., 1991; 1993; Babe et al., 1992).
The approach utilized in the second aspect of the invention takes advantage of the dimerization feature of PR and localizes a peptide corresponding to the dimer interface domain (The 4-amino acid sequence, TLNF as represented by the standard single-letter codes for amino acids) in the virus particle through chimeric Vpr. Our concept involves the incorporation of chimeric Vpr into the virus particle along with the Gag and Gag-Pol precursors where it is likely to interfere with the formation of active PR. The lack of availability of active PR will ultimately lead to the generation of immature, non-infectious virus particles due to incomplete processing of viral proteins. The data presented here shows that the presence of chimeric Vpr in viral particles indeed resulted in reduced levels of virus replication.